Role of skin punch biopsy in diagnosis of small fiber neuropathy-A review for the neuropathologist

Over the last three decades, skin punch biopsy has become the gold standard for diagnosis of small fiber neuropathies, including autonomic neuropathies commonly seen in diabetics, patients with HIV, and children with hereditary sensory autonomic neuropathies and toxin-induced neuropathy. Clinical, biochemical, electrophysiological tests are inconclusive, making it difficult to diagnose and initiate treatment. A skin punch biopsy is easy to perform in the outpatient clinic, is highly sensitive, and provides an objective diagnosis. Importantly, it helps avoid performing invasive nerve biopsy in patients with small fiber neuropathy, thereby preventing complications such as non-healing of the biopsy site, which is common in these patients. Secondly, the greatest advantage of skin punch biopsies is that they can be repeated any number of times, unlike a nerve biopsy, and are useful to evaluate disease progression and therapeutic response. More recently, its use has been expanded to the diagnosis of large fiber neuropathies, inherited demyelinating neuropathies, etc., obviating the need for a nerve biopsy. The European Federation of Neurological Societies has published guidelines for evaluation to ensure uniformity with regard to the site of biopsy, processing, and quantification. The evaluation of the skin biopsy involves morphometric assessment of the intraepidermal nerve fiber density using PGP 9.5 immunostained sections by bright-field microscopy. This review focuses on the practical aspects of skin punch biopsy and its utility for the practicing pathologist.

Conserved expression of ubiquitin carboxyl-terminal esterase L1 (UCHL1) in mammalian testes.

Spermatogonia, the adult germ cells that initiate spermatogenesis in mammalian testis, are capable of dividing both mitotically and meiotically. Isolation and preservation of spermatogonia helps in preserving genetic pool of endangered animals. In this context, identification of marker(s) that can distinguish spermatogonia from other cells in testis gains significance. Here, we examined the expression of ubiquitin carboxyl-terminal esterase L1 (UCHL1) gene and protein in the testes of several mammals, including highly endangered species. Semi-quantitative-reverse transcriptase-polymerase chain reaction (RT-PCR) analysis showed presence of UCHL1 amplicon of 442 bp in all the 18 mammals studied. Nucleotide sequence analysis of these amplicons and their predicted protein sequences revealed 88-99% and 95-100% homology with available human UCHL1 and UCHL1 sequences of other available species in the GenBank, respectively. Western blot analysis showed that UCHL1 protein size was unique in all wild mammals. Immunohistology results confirmed UCHL1 expression in the spermatogonia/gonocytes in testes of several mammals belonging to eight distinct families including highly endangered Felidae, Canidae and Cercopithecoidae. These findings suggest that UCHL1 expression is conserved in the mammalian testis, and could be used as a specific marker for gonocytes/spermatogonia for developing male germ-cell based conservation techniques.

A morphological study of vomeronasal organ of Korean black goat (Capra aegagrus hircus) / 대한수의학회지

The vomeronasal organ (VNO) plays an important role in reproduction and social activities in ruminants including goats. A morphological study on the structure of VNO and its epithelial cells was carried out in Korean black goats. Grossly, the VNO of Korean goats opens into mouth through incisive ducts. Microscopically, the epithelium of VNO consisted of medial sensory epithelium and lateral non-sensory epithelium. Several blood vessels and nerve bundles were observed in the lamina propria encased by vomeronasal cartilage. Immunohistochemical staining showed that protein gene product (PGP) 9.5 was immunostained in the receptor cells of the sensory epithelium and in some cells of the non-sensory epithelium. Galectin-3 was mainly observed in the supporting cells of sensory and non-sensory epithelium. Lectins including wheat germ agglutinin, Ulex europaeus agglutinin, Bandeiraea simplicifolia lectin Isolectin B4, Dolichos biflorus agglutinin and soybean agglutinin used in this study were bound in VNO sensory, non-sensory epithelia as well as in the lamina propria with varying intensity. Collectively, this is a first descriptive morphological study of VNO of Korean black goat with special reference to lectin histochemistry.

Cutaneous lesions sensory impairment recovery and nerve regeneration in leprosy patients

It is important to understand the mechanisms that enable peripheral neurons to regenerate after nerve injury in order to identify methods of improving this regeneration. Therefore, we studied nerve regeneration and sensory impairment recovery in the cutaneous lesions of leprosy patients (LPs) before and after treatment with multidrug therapy (MDT). The skin lesion sensory test results were compared to the histopathological and immunohistochemical protein gene product (PGP) 9.5 and the p75 nerve growth factor receptors (NGFr) findings. The cutaneous neural occupation ratio (CNOR) was evaluated for both neural markers. Thermal and pain sensations were the most frequently affected functions at the first visit and the most frequently recovered functions after MDT. The presence of a high cutaneous nerve damage index did not prevent the recovery of any type of sensory function. The CNOR was calculated for each biopsy, according to the presence of PGP and NGFr-immunostained fibres and it was not significantly different before or after the MDT. We observed a variable influence of MDT in the recovery from sensory impairment in the cutaneous lesions of LPs. Nociception and cold thermosensation were the most recovered sensations. The recovery of sensation in the skin lesions appeared to be associated with subsiding inflammation rather than with the regenerative activity of nerve fibres.

Effect of Probucol and Verapamil on Injury to Myocardium and Nerve Fibers in Rat Heart Induced by Doxorubicin

BACKGROUND: Doxorubicin is considered to be one of the most effective drugs to treat a variety of human cancers. However, the dose-dependent cardiotoxicity of doxorubicin limits its clinical usefulness. This study aimed to evaluate the effect of probucol and verapamil on the cardiac neurotoxicity and cardiomyopathy induced by the long-term use of doxorubicin. METHODS: Sprague-Dawley male rats were grouped as the control group, the doxorubicin treated group, the doxorubicin treated with probucol group, and the doxorubicin treated with verapamil group. The rats were treated for 4, 6, 8 and 10 weeks. H&E staining and immunohistochemical staining for protein gene product 9.5, caspase-3, heat shock protein 70, and hsp 25 were performed. RESULTS: The degree of interstitial inflammatory cell infiltration was mildest in the probucol treated group. The reduction in the number of nerve fibers in the probucol treated group was less than the other treatment groups. There was a negative correlation between the treatment duration and stained nerve fibers in all the treatment groups. The number of caspase-3 positive cells was more increased in the doxorubicin group and the verapamil treated group than in the control and probucol treated group. CONCLUSION: It is suggested that probucol partly contributed to the inhibition of doxorubicin-induced cardiac neurotoxicity and cardiomyopathy, whereas the verapamil had no effect.

The Morphological Study of the Protein Gene Product (PGP) 9.5-like Immunoreactive Enteric Nervous System in Murine Ileum: Close Relation to Interstitial Cells of Cajal (ICC) in Confocal Laser Scanning Microscopy / 대한해부학회지

This study was performed to investigate the morphology of the enteric nervous system and interstitial cells of Cajal (ICC) in the murine ileum. The PGP9.5-like immunoreactive (PGP9.5-LI) neurons and the c-Kit-like immunoreactive (c-Kit-LI) ICCs were stained by indirect immunofluorescence method and were observed under the confocal laser scanning microscopy. According to three dimensional reconstruction study, it was found that the PGP9.5-LI neurons and the c-Kit-LI ICCs were widely distributed in the intestinal wall : (1) In circular muscle layer, PGP9.5-LI nerve fibers were paralell to circular muscle layer. (2) In the myenteric plexus, the PGP9.5-LI nerve were closely apposed to the adjacent PGP9.5-LI nerve, constituting the networks. (3) In double-labeling immunohistochemistry using anti-PGP9.5 and anti-c-Kit antibodies, the c-Kit-LI networks encircled around the neural strands. The characteristic arrangement of the PGP9.5-LI enteric nervous system and the ICC containing c-Kit-positive cells provide a morphological basis upon the mechanism regulating gastro-intestinal motility and the pathogenesis of gatro-intestinal disorders.

Morphometric Evaluation of PGP9.5 and NCAM Expressing Nerve Fibers in Colonic Muscle of Patients with Hirschsprung's Disease

A quantitative assessment of the density of the protein gene product 9.5 (PGP9.5), the neural cell adhesion molecule (NCAM), and the low-affinity nerve growth factor receptor (NGFR) expressing nerve fibers in the circular muscle layer in the colon was carried out by morphometric analyses from 13 patients with Hirschsprung's disease (HD). The difference in the nerve fiber density between the ganglionic and aganglionic segments was compared by calculating the ratio of the sum of the areas occupied by positively stained nerve fibers per unit area of the muscle after immunohistochemical staining on paraffin embedded tissue sections using computer software. There was an obvious difference in the density of the PGP9.5 stained nerve fibers between the ganglionic (0.0380 +/- 0.0171) and aganglionic segments (0.0143 +/- 0.01661). The NCAM-positive nerve fibers were fewer in number than those of both the PGP9.5-positive fibers and NCAM-positive fibers, which were also markedly lower in number in the aganglionic segment (0.0066 +/- 0.0076) than in the ganglionic segment (0.0230 +/- 0.0195). Immunostaining for low-affinity NGFR revealed much fainter staining in the ganglionic and aganglionic segment without a statistically significant difference in their density. Considering the fact that PGP9.5 is a very sensitive marker for nerve fibers, the results of this study reaffirm the innervation failure of the proper muscle in HD. The decreased NCAM expression level in the aganglionic segment appears to be caused not by the selective down-regulation of NCAM expression among the nerve fibers but by a markedly reduced number of nerve fibers.

The Nerve-dependency of Merkel Cell Proliferation in Cultured Human Fetal Glabrous Skin

Merkel cells are thought to function as slowly adapting mechanoreceptors and are known as targets for sensory nerves. However, the nerve-dependency of Merkel cells remains controversial. In this respect, some investigators have found interregional differences between hairy and glabrous skin and others have shown intraregional differences within denervated rat touch domes. Differences between species have also been reported. This study was performed to determine whether Merkel cells proliferate in vitro in the absence of the systemic factors, blood vessels and the intact nerves in human skin. Suspension organ culture was performed using fetal digits to investigate their in vitro proliferation. Merkel cells and cutaneous nerves were identified using antibodies to cytokeratin 20 and protein gene product 9.5 (PGP 9.5), respectively. Fetal digits of 56-82 day gestational age were cultured in serum free medium in a high O2 (45%) environment. Tissues were harvested before starting culture (D0) and 1,4,7,14, 28d after culture. Merkel cells were observed in the volar pads and dorsal nail matrices at D0. After 28d of suspension organ culture, digits looked healthy structurally and the number of Merkel cells had increased. However, PGP 9.5-immunoreactive nerves were markedly diminished after 1 day of culture and almost disappeared after 4 days. Merkel cell proliferation in vitro suggested that Merkel cell development is probably nerve-independent in human fetal glabrous skin.

Estudo imunohistoquímico e ultra-estrutural da inervação de de lesões cutâneas de hanseníase em fase inicial / Immunohistochemistry and ultrastructural analysis of the innervation of the skin lesions of leprosy in early stage

A neuropatia hanseniana (NH) é a principal condição responsável pela incapacidade e deformidade apresentadas pelo paciente portador da hanseníase...O conhecimento aprofundado sobre a biologia da NH além de ser justificado pelo seu fascinante interesse biológico, faz-se necessário para os programas de controle que almejam não só a diminuição da prevalência e da incidência da hanseníase, mas também a redução da morbidade das lesões neurais incapacitantes causadas pela doença.